*Target (protein/gene name): Plasmodium falciparum 3D7- Glutathione Reductase
*NCBI Gene # or RefSeq#:811773 *Protein ID (NP or XP #) or Wolbachia#: Accession CZT99905.1 *Organism (including strain): Plasmodium falciparum 3D7 (isolate 3D7) Etiologic Risk Group (see link below): RG2 */Disease Information (sort of like the Intro to your Mini Research Write up): Plasmodium falciparum is the most deadly of the five Plasmodium species that cause human malaria. Malaria has a massive impact on human health; it is the worlds second biggest killer. Around 300 million clinical cases occur each year resulting in between 1.5 - 2.7 million deaths annually, with the majority of cases being present in sub-saharan Africa. It is estimated that 3,000 children under the age of five years fall victim to malaria each day. Additonally, around 40% of the worlds population are at risk. Link to TDR Targets page (if present): TDR Targets page Link to Gene Database page: NCBI Essentiality of this protein: Essential for glutathione metabolism and reductase pathway Is it a monomer or multimer as biological unit? (make prediction athttp://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): Complex of proteins?: No, Dimer formed from two identical monomers Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): Papers for druggable target *EC#: 1.8.1.7 Link to BRENDA EC# page: http://www.brenda-enzymes.org/enzyme.php?ecno=1.8.1.7
-- Show screenshot of BRENDA enzyme mechanism schematic Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): -- link to Sigma (or other company) page for assay (see Sigma links below) -- -or link (or citation) to paper that contains assay information http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Enzyme_Assay/glutathionereductase.pdf -- links to assay reagents (substrates) pages. http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Enzyme_Assay/glutathionereductase.pdf -- List cost and quantity of substrate reagents, supplier, and catalog #
100 mM Potassium Phosphate Buffer with 3.4 mM Ethylenediaminetetraacetic Acid
30 mM Glutathione Substrate Solution (GSSG)
0.8 mM ß-Nicotinamide Adenine Dinucleotide Phosphate, Reduced Form Solution
1.0% (w/v) Bovine Serum Albumin
Glutathione Reductase Enzyme Solution Structure (PDB or Homology model -- PDB # or closest PDB entry if using homology model: 3SQP for human and 1ONF for plasmodium Current Inhibitors:
(1R(S),2R(S),3S(R),4S(R))-2,3-dihydroxycyclo-hexane-1,4-diyl dinitrate Expression Information (has it been expressed in bacterial cells):
mutant enzyme glutathione reductase-trypanothine reductase, expression in Escherichia coli (Brenda) Purification Method:
ammonium sulfate fractionation, 2',5'-ADP Sepharose 4B affinity chromatography and Sephadex G-200 gel filtration (Brenda) Image of protein (PyMol with features delineated and shown separately):
Fig 1. Protein 1ONF PyMol image *Amino Acid Sequence (paste as text only - not as screenshot or as 'code'): MVYDLIVIGGGSGGMAAARRAARHNAKVALVEKSRLGGTCVNVGCVPKKIMFNAASVHDI LENSRHYGFDTKFSFNLPLLVERRDKYIQRLNNIYRQNLSKDKVDLYEGTASFLSENRIL IKGTKDNNNKDNGPLNEEILEGRNILIAVGNKPVFPPVKGIENTISSDEFFNIKESKKIG IVGSGYIAVELINVIKRLGIDSYIFARGNRILRKFDESVINVLENDMKKNNINIVTFADV VEIKKVSDKNLSIHLSDGRIYEHFDHVIYCVGRSPDTENLKLEKLNVETNNNYIVVDENQ RTSVNNIYAVGDCCMVKKSKEIEDLNLLKLYNEERYLNKKENVTEDIFYNVQLTPVAINA GRLLADRLFLKKTRKTNYKLIPTVIFSHPPIGTIGLSEEAAIQIYGKENVKIYESKFTNL FFSVYDIEPELKEKTYLKLVCVGKDELIKGLHIIGLNADEIVQGFAVALKMNATKKDFDE TIPIHPTAAEEFLTLQPWMKX
*length of your protein in Amino Acids: 500 aa Molecular Weight of your protein in kiloDaltons: 56.492 kDa Molar Extinction coefficient of your protein at 280 nm wavelength: 99115M^-1 cm^-1 TMpred graph Image
Fig 2. TMpred graph of the chains in glutathione reductase protein of Plasmodium Falciparum.
*CDS Gene Sequence (paste as text only): ATGGTTTACGACCTCATCGTTATCGGTGGTGGTTCTGGCGGTATGGCTGCGGCACGTCGT GCGGCTCGTCACAACGCGAAAGTTGCTCTGGTTGAAAAATCTCGTCTGGGCGGCACCTGC GTAAACGTAGGTTGCGTTCCGAAGAAGATCATGTTCAACGCGGCTAGCGTTCACGATATC CTGGAGAATTCCCGTCATTACGGTTTCGATACCAAATTCAGCTTTAACCTGCCGCTGCTC GTTGAGCGTCGTGACAAATACATCCAGCGTCTGAACAACATTTATCGTCAAAACCTCTCC AAAGACAAGGTTGACCTGTACGAGGGTACTGCGTCTTTCCTCTCCGAAAACCGCATTCTC ATCAAGGGTACGAAAGATAATAACAACAAGGACAACGGTCCGCTGAATGAGGAAATTCTG GAGGGCCGTAACATCCTGATCGCAGTAGGCAACAAGCCAGTTTTCCCGCCTGTGAAAGGT ATTGAGAACACCATCTCTTCTGATGAGTTCTTCAACATCAAAGAATCCAAAAAGATTGGC ATCGTTGGCAGCGGTTACATCGCGGTTGAGCTGATCAACGTGATCAAACGCCTCGGCATT GACTCTTACATTTTTGCGCGTGGCAACCGTATCCTGCGCAAATTCGACGAATCTGTGATT AATGTGCTCGAGAATGACATGAAAAAGAACAATATCAACATTGTGACCTTCGCGGACGTT GTTGAAATCAAGAAAGTTTCTGACAAGAACCTGAGCATTCACCTCAGCGACGGTCGTATC TACGAGCACTTCGACCACGTCATCTATTGCGTAGGTCGTTCCCCGGACACCGAAAATCTG AAACTGGAAAAACTGAACGTTGAGACCAACAATAACTACATTGTTGTCGACGAGAACCAG CGCACCTCTGTAAATAACATCTATGCAGTCGGTGACTGCTGCATGGTTAAGAAGTCTAAA GAGATCGAAGACCTGAACCTCCTGAAGCTGTATAACGAGGAGCGTTACCTCAACAAAAAG GAAAATGTTACCGAAGACATCTTCTATAACGTCCAGCTGACCCCGGTTGCTATCAATGCT GGTCGCCTCCTCGCGGATCGTCTGTTCCTCAAGAAGACCCGTAAAACCAACTACAAACTG ATCCCGACCGTTATTTTCTCCCACCCGCCGATTGGTACGATCGGTCTGTCCGAGGAAGCG GCGATCCAGATCTACGGTAAGGAGAACGTTAAGATCTATGAAAGCAAATTTACGAACCTG TTTTTCTCTGTTTATGACATCGAACCGGAACTGAAAGAAAAAACGTATCTCAAACTCGTG TGTGTTGGTAAAGACGAACTGATTAAAGGCCTGCACATCATCGGCCTGAATGCAGACGAA ATCGTTCAGGGTTTCGCCGTAGCGCTCAAAATGAACGCGACCAAGAAGGACTTCGATGAA ACCATCCCAATCCACCCAACTGCGGCGGAAGAGTTTCTCACTCTGCAGCCGTGGATGAAG TGA *GC% Content for gene (codon optimized):unable to to identify information
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
Primer design results for 'tail' primers (this is just 2 sequences):
Plasmodium falciparum 3D7- Glutathione Reductase
*Target (protein/gene name): Plasmodium falciparum 3D7- Glutathione Reductase
*NCBI Gene # or RefSeq#:811773
*Protein ID (NP or XP #) or Wolbachia#: Accession CZT99905.1
*Organism (including strain): Plasmodium falciparum 3D7 (isolate 3D7)
Etiologic Risk Group (see link below): RG2
*/Disease Information (sort of like the Intro to your Mini Research Write up):
Plasmodium falciparum is the most deadly of the five Plasmodium species that cause human malaria. Malaria has a massive impact on human health; it is the worlds second biggest killer. Around 300 million clinical cases occur each year resulting in between 1.5 - 2.7 million deaths annually, with the majority of cases being present in sub-saharan Africa. It is estimated that 3,000 children under the age of five years fall victim to malaria each day. Additonally, around 40% of the worlds population are at risk.
Link to TDR Targets page (if present): TDR Targets page
Link to Gene Database page: NCBI
Essentiality of this protein: Essential for glutathione metabolism and reductase pathway
Is it a monomer or multimer as biological unit? (make prediction at http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html):
Complex of proteins?: No, Dimer formed from two identical monomers
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): Papers for druggable target
*EC#: 1.8.1.7
Link to BRENDA EC# page: http://www.brenda-enzymes.org/enzyme.php?ecno=1.8.1.7
-- Show screenshot of BRENDA enzyme mechanism schematic
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
-- link to Sigma (or other company) page for assay (see Sigma links below)
-- -or link (or citation) to paper that contains assay information
http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Enzyme_Assay/glutathionereductase.pdf
-- links to assay reagents (substrates) pages.
http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Enzyme_Assay/glutathionereductase.pdf
-- List cost and quantity of substrate reagents, supplier, and catalog #
100 mM Potassium Phosphate Buffer with 3.4 mM Ethylenediaminetetraacetic Acid
30 mM Glutathione Substrate Solution (GSSG)
0.8 mM ß-Nicotinamide Adenine Dinucleotide Phosphate, Reduced Form Solution
1.0% (w/v) Bovine Serum Albumin
Glutathione Reductase Enzyme Solution
Structure (PDB or Homology model
-- PDB # or closest PDB entry if using homology model: 3SQP for human and 1ONF for plasmodium
Current Inhibitors:
(1R(S),2R(S),3S(R),4S(R))-2,3-dihydroxycyclo-hexane-1,4-diyl dinitrate
Expression Information (has it been expressed in bacterial cells):
mutant enzyme glutathione reductase-trypanothine reductase, expression in Escherichia coli (Brenda)
Purification Method:
ammonium sulfate fractionation, 2',5'-ADP Sepharose 4B affinity chromatography and Sephadex G-200 gel filtration (Brenda)
Image of protein (PyMol with features delineated and shown separately):
Fig 1. Protein 1ONF PyMol image
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MVYDLIVIGGGSGGMAAARRAARHNAKVALVEKSRLGGTCVNVGCVPKKIMFNAASVHDI LENSRHYGFDTKFSFNLPLLVERRDKYIQRLNNIYRQNLSKDKVDLYEGTASFLSENRIL IKGTKDNNNKDNGPLNEEILEGRNILIAVGNKPVFPPVKGIENTISSDEFFNIKESKKIG IVGSGYIAVELINVIKRLGIDSYIFARGNRILRKFDESVINVLENDMKKNNINIVTFADV VEIKKVSDKNLSIHLSDGRIYEHFDHVIYCVGRSPDTENLKLEKLNVETNNNYIVVDENQ RTSVNNIYAVGDCCMVKKSKEIEDLNLLKLYNEERYLNKKENVTEDIFYNVQLTPVAINA GRLLADRLFLKKTRKTNYKLIPTVIFSHPPIGTIGLSEEAAIQIYGKENVKIYESKFTNL FFSVYDIEPELKEKTYLKLVCVGKDELIKGLHIIGLNADEIVQGFAVALKMNATKKDFDE TIPIHPTAAEEFLTLQPWMKX
*length of your protein in Amino Acids: 500 aa
Molecular Weight of your protein in kiloDaltons: 56.492 kDa
Molar Extinction coefficient of your protein at 280 nm wavelength: 99115M^-1 cm^-1
TMpred graph Image
Fig 2. TMpred graph of the chains in glutathione reductase protein of Plasmodium Falciparum.
*CDS Gene Sequence (paste as text only):
ATGGTTTACGACCTCATCGTTATCGGTGGTGGTTCTGGCGGTATGGCTGCGGCACGTCGT GCGGCTCGTCACAACGCGAAAGTTGCTCTGGTTGAAAAATCTCGTCTGGGCGGCACCTGC GTAAACGTAGGTTGCGTTCCGAAGAAGATCATGTTCAACGCGGCTAGCGTTCACGATATC CTGGAGAATTCCCGTCATTACGGTTTCGATACCAAATTCAGCTTTAACCTGCCGCTGCTC GTTGAGCGTCGTGACAAATACATCCAGCGTCTGAACAACATTTATCGTCAAAACCTCTCC AAAGACAAGGTTGACCTGTACGAGGGTACTGCGTCTTTCCTCTCCGAAAACCGCATTCTC ATCAAGGGTACGAAAGATAATAACAACAAGGACAACGGTCCGCTGAATGAGGAAATTCTG GAGGGCCGTAACATCCTGATCGCAGTAGGCAACAAGCCAGTTTTCCCGCCTGTGAAAGGT ATTGAGAACACCATCTCTTCTGATGAGTTCTTCAACATCAAAGAATCCAAAAAGATTGGC ATCGTTGGCAGCGGTTACATCGCGGTTGAGCTGATCAACGTGATCAAACGCCTCGGCATT GACTCTTACATTTTTGCGCGTGGCAACCGTATCCTGCGCAAATTCGACGAATCTGTGATT AATGTGCTCGAGAATGACATGAAAAAGAACAATATCAACATTGTGACCTTCGCGGACGTT GTTGAAATCAAGAAAGTTTCTGACAAGAACCTGAGCATTCACCTCAGCGACGGTCGTATC TACGAGCACTTCGACCACGTCATCTATTGCGTAGGTCGTTCCCCGGACACCGAAAATCTG AAACTGGAAAAACTGAACGTTGAGACCAACAATAACTACATTGTTGTCGACGAGAACCAG CGCACCTCTGTAAATAACATCTATGCAGTCGGTGACTGCTGCATGGTTAAGAAGTCTAAA GAGATCGAAGACCTGAACCTCCTGAAGCTGTATAACGAGGAGCGTTACCTCAACAAAAAG GAAAATGTTACCGAAGACATCTTCTATAACGTCCAGCTGACCCCGGTTGCTATCAATGCT GGTCGCCTCCTCGCGGATCGTCTGTTCCTCAAGAAGACCCGTAAAACCAACTACAAACTG ATCCCGACCGTTATTTTCTCCCACCCGCCGATTGGTACGATCGGTCTGTCCGAGGAAGCG GCGATCCAGATCTACGGTAAGGAGAACGTTAAGATCTATGAAAGCAAATTTACGAACCTG TTTTTCTCTGTTTATGACATCGAACCGGAACTGAAAGAAAAAACGTATCTCAAACTCGTG TGTGTTGGTAAAGACGAACTGATTAAAGGCCTGCACATCATCGGCCTGAATGCAGACGAA ATCGTTCAGGGTTTCGCCGTAGCGCTCAAAATGAACGCGACCAAGAAGGACTTCGATGAA ACCATCCCAATCCACCCAACTGCGGCGGAAGAGTTTCTCACTCTGCAGCCGTGGATGAAG
TGA
*GC% Content for gene (codon optimized):unable to to identify information
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
Primer design results for 'tail' primers (this is just 2 sequences):
Tail TACTTCCAATCC ATG (3-5)
Start codon: (ATG)
Start bps GTTTACGATCTCATCGTTATCGGTGGC
3-5 BEGINNING
TACTTCCAATCC ATG GTTTACGATCTCATCGTTATCGGTGGC (27)
End bps CTGACTCTGCAACCGTGGATGAAG
Stop codon:TTA
Tails TATCCACCTTTACTG (3-5)
5-3 DIRECTION REVERSE COMPLEMENT
TATCCACCTTTACTG TTA CTTCATCCACGGTTGCAGAGTCAG (24)
Tail TACTTCCAATCC ATG (3-5)
Start codon: (ATG)
Start bps GTTTACGATCTCATCGTTATCGGTGGC
3-5 BEGINNING
TACTTCCAATCC ATG GTTTACGATCTCATCGTTATCGGTGGC (27)
End bps CTGACTCTGCAACCGTGGATGAAG
Stop codon:TTA
Tails TATCCACCTTTACTG (3-5)
5-3 DIRECTION REVERSE COMPLEMENT
TATCCACCTTTACTG TTA CTTCATCCACGGTTGCAGAGTCAG (24)
0.4Um Oligo conc, 50Mm Na+, 0.3Mm Dntp
forward with 27 start bp; 42 length; GC 45.2%
0mM: 65.4; 1.5mM: 73; 2mM 73.3; 4mM: 74mM; 6mM: 74.4
reverse with 24 ending bp; 42 length; GC 45.2%
0mM: 65.2; 1.5mM: 72.9; 2mM 73.2; 4mM: 73.9mM; 6mM: 74.2